Many experimental techniques have been applied to study the physiology of the nervous system. Several of those techniques are described below. One of the applications for which the development of such techniques is essential is to assist a surgeon during surgery to avoid or reduce damage to functional neuronal tissue. Techniques currently used for diagnostic and intraoperation assessment are also described below.
Hill and Keynes observed that the nerve from the walking leg of the shore crab (Carcinus maenas) normally has a whitish opacity caused by light scattering, and that opacity changes evoked by electrical stimulation of that nerve were measurable. Hill, D. K. and Keynes, R. D., "Opacity Changes in Stimulated Nerve," J. Physiol. 108:278-281 (1949). Since the publication of those results, experiments designed to learn more about the physiological mechanisms underlying the correlation between optical and electrical properties of neuronal tissue and to develop improved techniques for detecting and recording activity-evoked optical changes have been ongoing.
Several types of phenomenon relating to physiological neuronal activity have been detected. Thermographic studies have detected thermal radiation changes that take place during neuronal activation using infrared imaging techniques. Spectrophotometric techniques have been used to detect changes in absorption of the oxidizable fraction of cytochrome oxidase in brain tissue. Spectroscopic techniques such as electron microscopy and x-ray diffraction are not well-suited to studying physiological activity in living neuronal tissue because of the high risk of tissue damage.
Many biomolecules fluoresce as a result of excitation with emr at the wavelength of the molecule's absorption band. This excitation causes the molecule to emit part of the absorbed energy at a different wavelength, and the emission can be detected using fluorometric techniques. Most physiological studies measuring intrinsic fluorescence have selected for NADH, which is an important intermediate in oxidative catabolism. Furthermore, NADH concentration in neuronal tissue is believed to be correlated with neuronal activity. Upon excitation with ultraviolet light, NADH fluoresces at about 460 nm. Unfortunately, this technique would not be suitable for monitoring neuronal activity in humans, because illumination of in vivo neuronal tissue in vivo with ultraviolet light may cause serious tissue damage.
Another technique for detecting neuronal activity involves administration of a voltage-sensitive dye, whose optical properties change during changes in electrical activity of neuronal cells. The spatial resolution achieved by this technique is near the single cell level. For example, researchers have used the voltage-sensitive dye merocyanine oxazolone to map cortical function in a monkey model. Blasdel, G. G. and Salama, G., "Voltage Sensitive Dyes Reveal a Modular Organization Monkey Striate Cortex," Nature 321:579-585, 1986. However, the use of these kinds of dyes would pose too great a risk for use in humans in view of their toxicity. Furthermore, such dyes are bleached by light and must be infused frequently.
Intrinsic changes in optical properties of cortical tissue have been assessed by reflection measurements of tissue in response to electrical or metabolic activity. Grinvald, A., et al., "Functional Architecture of Cortex Revealed by Optical Imaging of Intrinsic Signals," Nature 324:361-364, 1986. Grinvald, et al., "Optical Imaging of Neuronal Activity", Physiological Reviews, Vol. 68, No. 4, October 1988. Grinvald and his colleagues reported that some slow signals from hippocampal slices could be imaged using a CCD camera without signal averaging.
A CCD camera was used to detect intrinsic signals in a monkey model. Ts'o, D. Y., et al., "Functional Organization of Primate Visual Cortex Revealed by High Resolution Optical Imaging," Science 249:417-420, 1990. The technique employed by Ts'o et al. would not be practical for human clinical use, since imaging of intrinsic signals was achieved by implanting a stainless steel optical chamber in the skull of a monkey and contacting the cortical tissue with an optical oil. Furthermore, in order to achieve sufficient signal to noise ratios, Ts'o et al. had to average images over periods of time greater than 30 minutes per image.
Optically imaging neuronal and other types of tissue using techniques and apparatus similar to those described herein is described in U.S. Pat. Nos. 5,215,095, 5,465,718, and 5,438,989, which are incorporated herein by reference in their entirety.
The mechanisms responsible for intrinsic signals are not well understood. Possible sources of intrinsic signals include dilation of small blood vessels, neuronal activity-dependent release of potassium, and swelling of neurons and/or glial cells caused, for example, by ion fluxes or osmotic activity. Light having a wavelength in the range of 500 to 700 nm may also be reflected differently between active and quiescent tissue due to increased blood flow into regions of higher neuronal activity. Yet another factor which may contribute to intrinsic signals is a change in the ratio of oxyhemoglobin and deoxyhemoglobin in blood.
One of the important applications for quantitative techniques that identify and assess neuronal tissue and function, both in the central and the peripheral nervous system, is to provide information to medical professionals prior to and during surgery. A neurosurgeon attempts to map boundaries of dysfunctional tissue, so that dysfunctional tissue is removed without affecting the surrounding tissue, and as much neuronal function as is possible is preserved. Neurological surgery is especially risky, and precise resection of dysfunctional tissue without removing functional tissue is critical. It is also important for surgeons working outside the central nervous system to locate peripheral nerves and avoid damaging them during other types of surgical procedures.
Current intraoperative techniques do not provide rapid or high resolution differentiation of dysfunctional neuronal tissue from normal neuronal tissue, or of neuronal tissue from surrounding tissue. Presently, electroencephalography (EEG) and electrocorticography (ECoG) techniques are used prior to and during neurosurgery for the purposes of identifying areas of abnormal neuronal activity. These measurements provide a direct measurement of the electrical activity in neuronal tissue.
One type of neurosurgical procedure which exemplifies these principles is the surgical treatment of intractable epilepsy (that is, epilepsy which cannot be controlled with medications). EEG and ECoG techniques are typically used to identify epileptic foci. Intraoperative EEG techniques involve placing an array of electrodes upon the surface of the cortex to detect electrical activity. This is done in an attempt to localize abnormal cortical activity of epileptic seizure discharge.
Although EEG techniques are of widespread use, hazards and limitations are associated with these techniques. The size of the electrode surface and the distance between electrodes in an EEG array are large with respect to the size of brain cells (e.g., neurons) with epileptic foci. Thus, current techniques provide poor spatial resolution (approximately 1.0 cm) of the areas of abnormal cortical activity. Further, EEG techniques do not provide a map of normal cortical function in response to external stimuli (such as being able to identify a cortical area dedicated to speech function by recording electrical activity while the patient speaks). A modification of this technique, called cortical evoked potentials, can provide some functional cortical mapping. However, the cortical evoked potential technique suffers from the same spatial resolution problems as the EEG technique.
The most common method of intraoperative localization of cortical function during neurosurgery is direct electrical stimulation of the cortical surface with a stimulating electrode. Using this technique, the surgeon attempts to evoke either an observed motor response from specific parts of the body, or in the case of an awake patient, to generate specific sensations or cause an interruption in the patient's speech output. Again, this technique suffers from the same problems as the EEG technique because it offers only crude spatial localization of function.
Possible consequences of the inaccuracies of all these techniques when employed, for example, to identify the portion of the cortex responsible for epileptic seizures, are either that a greater than necessary amount of cortical tissue is removed, possibly leaving the patient with a deficit in function, or that not enough tissue is removed, leaving the patient uncured by the surgery. Despite these inadequacies, such techniques have been deemed acceptable treatment for intractable epilepsy.
A need in the art remains for methods and apparatus for optically imaging neuronal tissue which can precisely and quickly distinguish functional and dysfunctional (e.g., viable and nonviable) neuronal tissue, distinguish neuronal tissue from surrounding non-neuronal tissue, and map cortical neuronal function. Quantitative techniques providing the following capabilities would be desirable for assessing neuronal tissue: the ability to provide electrophysiological information with a high degree of spatial and temporal resolution; the ability to monitor the activity of single neurons, as well as patterns of activity in larger areas of neuronal tissue, and the property of being physiologically non-invasive, i.e., providing data without requiring application of chemicals or penetration of mechanical devices, such as neuroelectrodes.